Abstract The Human Immunodeficiency Virus Type 1 (HIV-1, hereafter referred to as HIV) currently infects ~37 million people worldwide, and the number of infected individuals continues to rise. In the absence of a cure, antiretroviral therapy (ART) represents the primary treatment option, as it slows disease progression, and limits new infections. Integrase (IN) Strand Transfer Inhibitors (INSTIs) are a class of ART that block integration of viral DNA into host chromosomes, a process that is mediated by the viral IN enzyme, which assembles into oligomeric nucleoprotein complexes on the ends of viral DNA, termed ?intasomes?. INSTIs selectively target intasomes and represent first-line therapies in the clinic. However, the emergence of IN variants resistant to INSTIs is a major clinical problem. Structural biology approaches can decipher the molecular mechanisms underlying drug action and resistance, providing useful information for rationally improving current therapies. In this proposal, approaches centered around revolutionary advances in cryo-electron microscopy for structural studies will be used to understand how INSTIs interact with their natural drug target, the HIV intasome, as well as mechanisms by which resistance to these drugs emerges. The proposed aims will address several major themes. Aim 1 will define the mechanisms of action of clinical and developmental INSTIs in the context of HIV intasomes. Procedures used to perform high-resolution structural studies of INSTI-bound complexes by cryo-EM will then be adopted to decipher novel, clinically relevant mechanisms of drug resistance that arise in response to INSTI treatment. This work will be complemented using biochemical and virology assays designed to dissect key interactions between HIV intasomes and INSTIs and to validate structural findings. Aim 2 will extend these findings to define INSTI mechanism of action in the presence of biologically relevant cellular factors, including methylated mononucleosomes (mMNs; the natural target for HIV integration) and lens epithelium-derived growth factor (LEDGF). Aim 2 will therefore define the biochemical and structural mechanisms by which INSTIs interact with and inhibit IN catalytic activity in the context of the intasome-LEDGF-mMN complex, thereby elucidating how INSTIs function in infected cells and their precise stage of activity. In addition to providing the first structural information for INSTI interactions with their natural drug target in the presence of relevant cellular factors, this work will: 1) elucidate how mutations within the IN active site disrupt drug binding, 2) define the precise stage and mechanism of action of this important class of drugs in a cellular context, and 3) provide blueprints for the rational improvement of future INSTIs. !